EP0186379B1 - Gen für ein Protein mit insektizider Wirkung - Google Patents

Gen für ein Protein mit insektizider Wirkung Download PDF

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EP0186379B1
EP0186379B1 EP85308994A EP85308994A EP0186379B1 EP 0186379 B1 EP0186379 B1 EP 0186379B1 EP 85308994 A EP85308994 A EP 85308994A EP 85308994 A EP85308994 A EP 85308994A EP 0186379 B1 EP0186379 B1 EP 0186379B1
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plasmid
dna fragment
amino acid
protein
insecticidal
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EP0186379A1 (de
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Yuji C/O Suntory Limited Shibano
Teruo C/O Suntory Limited Amachi
Hajime C/O Suntory Ltd. Yoshizumi
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Suntory Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins

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  • the present invention relates to genes coding for insecticidal proteins, plasmids containing the genes, and microorganisms transformed with the plasmids.
  • insecticidal protein produced by Bacillus thuringiensis shows toxicity against larvae of Lepidoptera
  • preparations containing the insecticidal protein have been commercially provided as insecticides with selective toxicity to insects.
  • insecticidal protein is produced only during the sporulation period, by Bacillus thuringiensis, the production period is rather short, and this limits the productivity of the protein. Therefore, the microbial insecticide is higher in cost than conventional chemically synthesized insecticides.
  • the insecticidal protein produced by Bacillus thuringiensis is characteristically crystalline, and has a ooor solubility in water. Accordingly, it is difficult to economically separate and purify the protein from the spores of producer cells. Therefore, the conventional microbial insecticides are prepared as formulations containing spores of Bacillus thuringiensis, which spores will pollute the environment when the formulation is used.
  • JP-A-58-500565 (equivalent to EP-A-63949 and WO 82/03872), refers to a DNA fragment coding for an insecticidal protein of Bacillus thuringiensis subsp. sotto origin, but does not include a detailed description thereof. Moreover, the molecular weights of a plasmid included in the Bacillus thuringiensis subsp. sotto, and of the produced insecticidal protein, are different from those of the present invention.
  • the invention provides a DNA fragment comprising a nucleotide sequence coding for an insecticidal protein having an amino acid sequence as set out in Fig. 1, or having an amino acid sequence comprising at least 723 amino acids from the N-terminus thereof.
  • the invention provides a DNA fragment having the nucleotide sequence from position 153 to position 3692 (codons 1 to 1180) as set out in Fig. 1, or at least 723 codons from the 5'- terminus of that sequence.
  • the invention provides a plasmid capable of replicating in E. coli and expressing the insecticidal protein defined above, comprising a pBR322 plasmid in which a DNA sequence as defined has been inserted.
  • the invention provides E. coli transformed with the plasmid so as to produce insecticidal protein.
  • the invention provides water-soluble insecticidal protein produced by an organism transformed with said DNA.
  • the invention provides processes comprising the production of the DNA fragments, plasmids and proteins described.
  • the DNA fragment coding for an insecticidal protein according to the present invention is derived from a plasmid pSY1 present in Bacillus thuringiensis subsp. sotto and including a gene for insecticidal protein.
  • the plasmid pSY1 is isolated from the above-mentioned strain according to a modification of the method described by Hansen and Olsen, J. Bacteriol. 135, 227-238 (1978) .
  • the molecular size of the plasmid is calculated to be 68 kb.
  • the plasmid pSY1 is cleaved with a restriction enzyme Xhol to obtain a DNA fragment containing a gene coding for insecticidal'protein.
  • the DNA fragment is then inserted into an Xhol site of a cloning vector pKAT 1
  • the ligation mixture is used to transform E. coli C600, and the transformants are screened for ampicillin resistance and the production of insecticidal proteins as determined by radioimmunoassay to obtain a clone containing plasmid pSX8, which includes an 18 kb insert containing a nucleotide sequence coding for the insecticidal protein.
  • the plasmid pSX8 is cleaved with various kinds of restriction enzymes, and each of the DNA fragments thus obtained is ligated to a cloning vector pBR322 cleaved with the corresponding restriction enzyme.
  • Each of the ligation mixtures is used to transform E. coli HB101, and the transformants are screened as described above to obtain a clone E. coli HB101/pSP801, which contains a plasmid pSP801 of about 11.0 kb including a 6.6 kb Pstl DNA insert.
  • a restriction map of the plasmid pSP801 is represented in Fig. 4.
  • E. coli HB101-pSP801 produces a polypeptide having a molecular weight of about 144 kilo Daltons (kD), and this molecular weight is approximately the same as that of a crystal protein produced by Bacillus thuringiensis, the insert of about 6.6 kb in the plasmid pSP801 is supposed to contain a nucleotide sequence coding for the entire amino acid sequence of a protein, the same as the crystal protein produced by Bacillus thuringiensis.
  • the plasmid pSP801 is then cleaved with restriction enzymes M1 ul and Hpal to obtain a DNA fragment of about 2.95 kb, which is then filled-in, and added with Bam HI linker at both terminals.
  • the DNA fragment thus modified is then inserted into a Bam HI site of pBR322 to obtain a ligation mixture.
  • the mixture is used to transform E. coli HB101, and the transformants are screened as described above to obtain a clone E. coli HB101/pLBT291.
  • the clone E. coli HB101 pLBT291 produces a polypeptide of about 75 kD having insecticidal activity. Therefore, the above-mentioned DNA fragment of about 2.95 kb is confirmed to contain a nucleotide sequence coding for a part of the amino acid sequence of the insecticidal crystal protein produced by Bacillus thuringiensis.
  • a nucleotide sequence of the DNA fragment of about 2.95 kb corresponds to a nucleotide sequence at positions 1 to 2955 in Fig. 1-1 to 1-7.
  • the DNA fragment consists of 2955 bases, in which 2802 bases from the positions 153 to 2954 encode 934 amino acids corresponding to the above-mentioned polypeptide.
  • Another part of the DNA fragment consisting of nucleotides from the positions 1 to 152 is a 5' non-coding sequence.
  • the nucleotide sequence is different from the nucleotide sequence disclosed by Wong et. al., supra , at three sites, which difference results in a difference of three amino acids in the peptide.
  • Plasmid pSY1 was prepared from Bacillus thuringiensis subsp. sotto according to a modification of the alkali-method described by Hansen and Olsen, J. Bacteriol. 135, 227-238 (1978).
  • the above-mentioned bacterium was cultured in 400 ml of Penassay broth, and the cultured cells were suspended in 10.8 ml of a solution comprised of 25% sucrose, 50 mM Tris-HCI, and 20 mM EDTA (pH 8.0), and the suspension was added with 10 mg/ml of lisozyme and incubated at 37° C for one hour.
  • the suspension was then added with pronase E (T.M) and sodium dodecyl sulfate (SDS) to concentrations of 0.5 mg/ml and 1% respectively, and incubated at the same temperature for another one hour to lyse the cells completely.
  • the lysate was added with 3 ml of 3N NaOH and incubated at a room temperature for three minutes, and then added with 8 ml of 2M Tris-HCI (pH 7.0), 4 ml of 20% SDS, 8 ml of 5M NaCI, and incubated for six hours in ice.
  • the thus-treated lysate was centrifuged at 17,000 x G for 30 minutes to obtain a supernatant.
  • polyethylene glycol 6000 was added to a concentration of 10% to precipitate DNA.
  • DNA was dissolved in 20 ml of a solution comprised of 20 mM Tris-HCI, 5 mM EDTA and 150 mM NaCl (ph 7.5), and the solution was added with RNase A to a concentration of 0.1 mg/ml, incubated at 37° C for two hours, extracted with phenol, and added with two volumes of ethanol to precipitate DNA.
  • the precipitate was dissolved in 5 ml of a solution comprising 20 mM Tris-HCI, 5 mM EDTA, and 150 mM NaCl (pH 7.5), and the solution was passed through a Biogel A-0.5 m (T.M.) column, and a void volume fraction was recovered.
  • DNA of plasmid pSY1 was treated with restriction enzymes.
  • the DNA was cleaved with Bam HI and Stu I at one site, with Bgl II and Xho I at three sites, and with Mlu I and Sac I at four sites, but not cleaved with Aat II, Sal I and Sma I.
  • Bon I, Bcl II, Cla I, Eco RI, Hind III, Hpa I, Kpn I, Pst I and Pvu II provided many fragments.
  • Figure 2 represents a restriction map obtained by Bgl II and Xho I.
  • Cleavage sites of Bam HI and Stu I locate on a Bgl II-A fragment and an Xho I-A fragment, respectively, at a distance of about 2 kb.
  • the plasmid pSYI was found to be 68 kb in size.
  • E. coli transformed with a plasmid containing a gene for an insecticidal protein were selected according to modification of the procedure described by Erlich et. al., Cell, 13 , 681-689 (1978) using an antibody against the insecticidal protein.
  • Bacillus thuringiensis subsp. sotto was cultured in a modified G medium described by Aronson et. al., J. Bacteriol. 106, 1016-1025 (1971) for three days, and the insecticidal crystal protein was isolated and purified from the culture according to a method of Sharpe et. al., Appl. Microbiol. 30, 1052-1053 (1975). The purified protein was used as an antigen to prepare the antibody.
  • the antibody IgG thus obtained was labeled with 125, according to a procedure described by Marrison and Bayse, Biochemistry 9 , 2995-3000 (1970).
  • Cells of E. coli to be tested were cultured on an agar plate medium to form colonies, and the agar plate carrying the colonies thereon was exposed to vapor of chloroform for 30 minutes, and overlaid with an agar layer containing 1 mg/ml lysozyme, 10 mM EDTA and 0.02% Triton X100 (T.M) to lyse cells of the colonies.
  • T.M Triton X100
  • the filter paper was washed with 10 mM phosphate buffer pH 7.2 containing 5% bovine serum and 0.15M NaCl (CS-PBS), and maintained in 10 mN Tris-HCI buffer pH 7.5 containing 0.14M NaCI, 25% bovine serum and 4 x 10 6 cpm 125 I-labeled antibody prepared as described above for 5 to 7 hours.
  • the filter was then washed with CS-PBS, dried, and exposed on an X-ray film to carry out autoradiography.
  • plasmid pSX8 The route for the construction of plasmid pSX8 is shown in Fig. 3.
  • the plasmid pSY1 was cleaved with a restriction enzyme Xhol to obtain DNA fragments.
  • a cloning vector plasmid pKAT1 was cleaved with Xho I.
  • the DNA fragments from pSY1 were then inserted to Xho site of the cleaved pKAT1 with T4 DNA ligase to obtain a ligation mixture, which was then used to transform cells of E. coli C600.
  • the transformants were first screened on L broth agar plate medium supplemented with 25 ⁇ g/ml ampicillin to select clones resistant to ampicillin, the selected clones were then subjected to the radioimmunoassay described in paragraph (3), and a positive clone containing recombinant plasmid pSX8 was selected.
  • the plasmid pSX8 contains a 18 kb insert of DNA fragment, which includes a gene coding for an insecticidal protein.
  • the plasmid pSX8 was cleaved with a restriction enzyme Hind III, Pst 1, or Eco RI to obtain DNA fragments, which were then inserted into a corresponding restriction site of cloning vectors pBR322 and pBR325 cleaved with the corresponding restriction enzyme, and each ligation mixture was used to transform E. coli HB101.
  • the transformants thus obtained were then screened according to the above- described procedures to select a clone expressing an insecticidal protein.
  • a clone E from the ligation of Pst I - cleaved pSX8 DNA fragment with Pst I - cleaved pBR322 fragment, a clone E.
  • coli HB101/pSP801 containing plasmid pSP801 was selected.
  • the route for construction of the plasmid pSP801 is shown in Fig. 3, and a restriction map of the plasmid pSP801 is shown in Fig. 4.
  • the plasmid pSP801 contains an inserted Pst I DNA fragment of 6.6 kb, which includes a gene coding for an insecticidal protein.
  • plasmid pSP801 was cleaved with Mlu I and Hpa I, and the Mlu I - HPa I DNA fragment thus obtained was filled-in, and attached with Bam HI linkers at both terminals.
  • the Bam HI fragment was then inserted into a Bam HI site of pBR322.
  • the ligation mixture thus prepared was used to transform E. coli HB101.
  • the transformants were then screened according to the radioimmunoassay described in paragraph (3) to obtain a clone E. coli HB101 ' pLBT291, containing plasmid pLBT291.
  • the plasmid pLBT291 contained a 2.95 kb insert of DNA fragment which corresponds to the Hpa I ⁇ 2 - Mlu I fragment on plasmid pSP801.
  • deletion derivatives were constructed from pSP801 using the restriction sites indicated in Fig. 5. Each of these derivatives was used to transform E. coli HB101, and a clone containing plasmid pHP1, pMR4, pKA2, pKB5, or pKB53 was obtained.
  • plasmid pMR4 contained all regions of the 6.6 kb-Pstl DNA fragment and 4 base pairs insertion at the Mlu I site, but other plasmids contained only parts of the Pstl DNA fragment.
  • Fig. 5 the left part of the drawing represents profiles of a region of each plasmid corresponding to the 6.6-kb Pst I DNA fragment in plasmid pSP801.
  • the right side columns represent the results of the radioimmunoassay and bioassay respectively, wherein + denotes positive, - denotes negative, and + denotes intermediate activity. From, Fig. 5, it is confirmed that the gene coding for an aimed insecticidal protein is located on the 2.95 kb Hpa I ⁇ 2 - Mlu I fragment in pSP801.
  • the clones which were pronounced positive in the radioimmunoassay i.e., E. coli HB101/pSX8, E. coli HB 1 01/pS801, E. coli HB101/pLBT291, and other clones containing pKB5,-pKA2, pMR4 and pHP1 respectively, as well as the control clone, i.e., E.
  • coli HB101/pBR322 were cultured in 400 ml of L-broth medium supplemented with 25 ⁇ g/ml of ampicillin, overnight at 37° C, and the cultured cells were collected by centrifugation and suspended in a buffer solution containing 150 mM NaCl, 20 mM Tris-HCI (pH8.0), 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, and 7 mM mercaptoethanol. The suspension was added with 0.3 ml of 10 mg/ml lysozyme, maintained on ice for 30 minutes, and sonicated for three minutes to rupture the cells.
  • the sonicated product was then centrifuged at 14,000 xg for 20 minutes to recover a supernatant.
  • the supernatant was then dialysed overnight in 20 mM phosphate buffer pH7.2.
  • the extracts thus obtained from the cultured E. coli were subjected to electrophoresis and bioassay.
  • the electrophoresis the extracts were separated on 7% SDS-polyacrylamide gel according to a conventional procedure.
  • the separated products were electrophoretically transferred to a nitrocellulose filter, and the nitrocellulose filter was incubated successively with an antibody against the insecticidal crystalline protein, anti-rabbit IgG antibody labeled with peroxidase, and then with a substrate solution for the peroxidase.
  • lane 1 represents a product from a clone containing plasmid pLBT291
  • lane 2 represents that from pKB5
  • lane 3 represents that from pKA2
  • lane 4 represents that from pMR4
  • lane 5 represents that from pHP1
  • lane 6 represents that from pSP801
  • lane 7 represents that from pSX8, and
  • lane 8 represents that from pBR322.
  • the insecticidal protein produced by Bacillus thuringiensis has been known to comprise three polypeptides, i.e., a main polypeptide having a molecular weight of 144 kD, and two minor polypeptides having a molecular weight of 97 kD and 76 kD respectively
  • the plasmids pMR4 (lane 4), pHP1 (lane 5), pSP801 (lane 6), and pSX8 (lane 7) provided a band corresponding to 144 kD, which corresponded to the main polypeptide of the insecticidal protein produced by Bacillus thuringiensis.
  • plasmid pLBT291 provided a band corresponding to 75 kD.
  • Plasmids pKB5 and pKA3 which were not pronounced positive in the radioimmunoassay and bioassay, and the control plasmid pBR322, provided no clear bands.
  • the above-mentioned result shows that the Pst I fragment in pSP801 and in pMR4, and the Hpa 1.2 - Pst I fragment in pHP1 contain a DNA sequence coding for a full length polypeptide of 144 kD.
  • the Hpa 1.2 - Mlu I fragment of 2.95 kb in pLBT291 corresponds to a 5'-terminal side part of the Hpa 1.2 - Pst I fragment in pHP1
  • the Hpa 1.2 - Mlu I 2.95 kb fragment in pLBT291 should encode a polypeptide corresponding to a part of the 144 kD polypeptide.
  • both the 75 kD polypeptide derived from pLBT291 and the 144 kD polypeptide derived from other plasmids react with the antibody against the insecticidal protein obtained from Bacillus thurringiensis. This suggests that the 75 kD polypeptide contains an essential region or site for the insecticidal activity.
  • the 75 kD polypeptide expressed by pLBT291 shows an insecticidal activity substantially identical to that of the 144 kD polypeptide which corresponds to the main polypeptide of the insecticidal crystal protein produced by Bacillus thuringiensis. This means that the 75 kD polypeptide contains a site or region essential for the insecticidal activity. Moreover, it should be noted that, in contrast with the natural insecticidal crystal protein produced by Bacillus thuringiensis, which is almost insoluble in water, both the 144 kD polypeptide and 75 kD polypeptide expressed by genes of the present invention are soluble in water.
  • the sequence of the Hpa 1.2 - Mlu I DNA fragment of 2.95 kb in plasmid pSP801, corresponding to the Bam HI DNA fragment of 2.95 bp in plasmid pLBT291 was determined according to the dideoxy method described by Messing, Method in Enzymology Vol. 101 C, 20-78.
  • a detailed restriction map of the Hpa 1.2 - Mlu I fragment is set forth in Fig. 6.
  • the 2.95 kb DNA fragment was cleaved with restriction enzymes Sau 3A, Hpa II, and Taq I, and the resulting DNA fragments were cloned into phage M13mp10, or M13mp11. Moreover, the 2.95 kb DNA fragment was cleaved with restriction enzymes Cla I, Hind III, and Eco RI, and the resulting DNA fragments were cloned into phage M13mp10 or M13mp11. These cloned minute DNA fragments were sequenced according to the above-cited Messing's method, and the determined sequences were overlapped to determine a whole nucleotide sequence of the 2.95 kb DNA fragment.
  • the Mlu I - Pst I DNA fragment of 2 kb positioned adjacent to the 3'-terminal of the Hpa 1.2 - Mlu I 2.95 kb fragment was sequenced as above-mentioned. In this manner, finally, the whole nucleotide sequence of the 5 kb DNA fragment from the Hpa 1.2 site to the Pst I site was determined.
  • the nucleotide sequence thus determined is set forth in Fig. 1-1 to Fig. 1-10.
  • the 5 kb DNA fragment consists of 5,002 bp, and contains a single large open reading frame starting from a starting codon ATG, wherein base "A" corresponds to the position 153 in the sequence in Fig. 1-1, and terminating at a stop codon TAA, wherein the last base "A” corresponds to the position 3,695 in the sequence in Fig. 1-8.
  • the open reading frame consists of 3,540 bp, and is flanked by a 5'-non-translation sequence consisting of 152 bp, and by a 3'-flanking sequence consisting of 1,310 bp.
  • the 144 kD insecticidal polypeptide should be derived from the above-mentioned open reading frame.
  • the open reading frame consisting of 3,540 bp encodes 1180 amino acids. Therefore, the 144 kD insecticidal polypeptide is assumed to consist of 1180 amino acid residues starting from Met at the amino acid position 1 and terminating by Glu at the amino acid position 1180, which corresponds to 133,481 Dalton of the molecular weight. This amino acid sequence reveals that approximately half at the amino terminal side of the 144 kb insecticidal polypeptide contains much less lysine and cysteine than a carboxy terminal side does.
  • nucleotide sequence starting from base “A” at position 153 and terminated by "G” at the position 2954, consisting of 2802 bases corresponds to an open reading frame contained in the 2.95 kb Hpa 1.2 - Mlu I DNA fragment.
  • the nucleotide sequence consisting of 2802 bases should encode 934 amino acids. Therefore, the polypeptide produced of E. coli transformed with plasmid pLBT291 may consist of at least 934 amino acid residues starting from Met at the amino acid position 1 to Arg at the amino acid position 934.
  • An inverted repeat sequence capable of forming a stable stem and loop structure is found from the position 3782, which corresponds to about 100 bp downstream from the stop codon, to the position 3824, and assumed to act as a terminater on transcription. Moreover, an open reading frame reversely extending from the position 5002 to the position 4021 is found.
  • the insecticidal protein are provided in a water-soluble form. Therefore, the protein can be easily separated and purified from the producer cells. This means that insecticidal compositions specific to Lepidoptera can be produced in a form containing no producer microorganism's cells or spores. Such compositions, when used, are safe for the environment.
  • E. coli transformed with the present plasmid produces the insecticidal protein without the limitation in the production period, in contrast with Bacillus thuringiensis which produces it only within the spore forming period, according to the present invention, the insecticidal protein can be produced at a lower cost.
  • nucleotide sequences and plasmids of the present invention can be further manipulated or modified to construct improved nucleotide sequences or plasmids which will provide new polypeptides having stronger insecticidal activity.

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Claims (17)

1. DNA-Fragment, enthaltend eine für ein insektizides Peptid codierende Nucleotidsequenz mit der folgenden Aminosäuresequenz:
Figure imgb0038
Figure imgb0039
Figure imgb0040
Figure imgb0041
Figure imgb0042
oder eine Aminosäuresequenz, die mindestens 723 Aminosäuren von dem N-Endpunkt dieser Aminosäuresequenz aus enthält.
2. DNA-Fragment nach Anspruch 1, wobei die Nucleotidsequenz die folgende Sequenz enthält:
Figure imgb0043
Figure imgb0044
Figure imgb0045
Figure imgb0046
oder eine Sequenz, die mindestens 723 Codons von dem 5'-Endpunkt dieser Nucleotidsequenz aus enthält.
3. DNA-Fragment nach Anspruch 1 oder Anspruch 2, mit einer Länge von etwa 6,6 kb.
4. DNA-Fragment nach Anspruch 1, wobei die durch die Nucleotidsequenz codierte Aminosäuresequenz eine Aminosäuresequenz, beginnend mit Met an der Aminosäureposition 1 und beendet bei Arg an der Aminosäureposition 934, wie in Anspruch 1 definiert, enthält.
5. DNA-Fragment nach Anspruch 4, wobei die für die Aminosäuresequenz codierende Nucleotidsequenz eine Nucleotidsequenz, beginnend mit Position 153 und beendet an Position 2955, wie in Anspruch 2 definiert, enthält.
6. DNA-Fragment nach einem der Ansprüche 1, 4 und 6, mit einer Länge von etwa 2,95 kb.
7. Plasmid, das zum Reduplizieren in E. coli und zur Expression des in Anspruch 1 definierten Peptids befähigt ist, enthaltend ein Plasmid pBR322, bei den die in Anspruch 1 definierte DNA-Sequenz eingefügt ist.
8. Plasmid nach Anspruch 7, wobei das eingefügte DNA-Fragment eine Länge von etwa 6,6 kb besitzt.
9. Plasmid nach Anspruch 8, wobei das DNA-Fragment an der Pst I-Aufbrechungsstelle in das Plasmid pBR322 eingefügt ist.
10. Plasmid nach Anspruch 7, wobei das eingefügte DNA-Fragment eine Länge von etwa 2,95 kb besitzt.
11. Plasmid nach Anspruch 10, wobei das DNA-Fragment an der Bam HI-Aufbrechungsstelle in das Plasmid pBR322 eingefügt ist.
12. Mit einem Plasmid nach einem der Ansprüche 7 bis 11 transformierte und ein insektizides Protein erzeugende E. coli.
13. E. coli nach Anspruch 12, wobei das Protein ein Molekulargewicht von 144 kD hat.
14. E. coli nach Anspruch 12, wobei das Protein ein Molekulargewicht von 75 kD hat.
15. Insektizides Protein, das durch einen derart transformierten Organismus erzeugt ist, daß ein DNA-Fragment nach einem der Ansprüche 1 bis 6 eingebracht ist.
16. Zusammensetzung, enthaltend ein Protein nach Anspruch 15 in insektizid wirksamer Menge, zusammen mit einem Trägerstoff.
17. Verfahren zur Herstellung eines insektiziden Proteins, umfassend die Herstellung des Proteins durch einen Organismus, der derart transformiert wird, daß ein DNA-Fragment nach einem der Ansprüche 1 bis 6 eingebracht wird.
EP85308994A 1984-12-12 1985-12-11 Gen für ein Protein mit insektizider Wirkung Expired - Lifetime EP0186379B1 (de)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0224331A1 (de) * 1985-10-28 1987-06-03 Sumitomo Chemical Company, Limited Herstellung von Insektizidprotein von Bacillus thuringiensis subsp. aizawai IPL durch die Expression von Insektizidprotein-Gen in Wirtszellen
FR2627778A1 (fr) * 1988-02-25 1989-09-01 Sandoz Sa Gene de structure, proteine endotoxine correspondante et application insecticide
EP0366397A1 (de) * 1988-10-27 1990-05-02 Mycogen Corporation Bacillus thuringiensis, benannt B.t. ps81f, welches aktiv gegen Lepidoptera ist, sowie Gen, welches für das gegen Lepidoptera aktive Toxin kodiert
EP0367474A1 (de) * 1988-11-01 1990-05-09 Mycogen Corporation Bacillus thuringiensis, benannt B.t. ps81gg, welches aktiv gegen Lepidoptera ist, sowie Gen welches für das gegen Lepidoptera aktive Toxin kodiert
EP0451878A1 (de) * 1985-01-18 1991-10-16 Plant Genetic Systems, N.V. Modifizierung von Pflanzen mittels gentechnologischer Verfahren, um Insekten zu bekämpfen oder zu kontrollieren
US5231008A (en) * 1985-10-28 1993-07-27 Sumitomo Chemical Company, Limited Production of insecticidal protein of bacillus thuringiensis subsp. aizawal IPL by the expression of insecticidal protein gene in host cells
US5317096A (en) * 1985-01-18 1994-05-31 Plant Genetic Systems, N.V. Transformation vectors allowing expression of foreign polypeptide endotoxins from Bacillus thuringiensis in plants
US5608142A (en) * 1986-12-03 1997-03-04 Agracetus, Inc. Insecticidal cotton plants
USRE35714E (en) * 1985-10-28 1998-01-13 Sumitomo Chemical Company, Limited Production of insecticidal protein of Bacillus thuringiensis subsp. aizawai by the expression of insecticidal protein gene in host cells

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JPS63133999A (ja) * 1986-11-27 1988-06-06 Agency Of Ind Science & Technol 誘導発現による蛋白質の製造法

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WO1982003872A1 (en) 1981-04-27 1982-11-11 Washington Univ B.thuringiensis crystal protein in e.coli
FR2525630B1 (fr) * 1982-04-26 1985-07-05 Pasteur Institut Adn contenant une sequence codant pour une proteine du cristal ou un polypeptide doue de proprietes insecticides, micro-organismes transformes par de tels adn, compositions contenant lesdites proteines du cristal, polypeptide ou micro-organismes
CA1301094C (en) * 1984-08-31 1992-05-19 Helen Riaboff Whiteley Bacillus thuringiensis crystal protein gene toxin segment
US7286984B1 (en) 1999-11-05 2007-10-23 At&T Corp. Method and system for automatically detecting morphemes in a task classification system using lattices

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US6107546A (en) * 1985-01-18 2000-08-22 Aventis Cropscience N.V. Transformation vectors allowing expression of truncated BT2 endotoxins in plants
US5843898A (en) * 1985-01-18 1998-12-01 Plant Genetic Systems, N.V. Transformation vectors allowing expression of foreign polypeptide endotoxins in plants
US5767372A (en) * 1985-01-18 1998-06-16 Plant Genetic Systems, N.V. Transformation vectors allowing expression of foreign polypeptide endotoxins from Bacillus thuringiensis in plants
US5760181A (en) * 1985-01-18 1998-06-02 Plant Genetic Systems, N.V. Endotoxins
EP0451878A1 (de) * 1985-01-18 1991-10-16 Plant Genetic Systems, N.V. Modifizierung von Pflanzen mittels gentechnologischer Verfahren, um Insekten zu bekämpfen oder zu kontrollieren
US5317096A (en) * 1985-01-18 1994-05-31 Plant Genetic Systems, N.V. Transformation vectors allowing expression of foreign polypeptide endotoxins from Bacillus thuringiensis in plants
US5545565A (en) * 1985-01-18 1996-08-13 Plant Genetic Systems, N.V. Transformation vectors allowing expression of foreign polypeptide endoxins from Bacillus thuringiensis in plants
USRE35714E (en) * 1985-10-28 1998-01-13 Sumitomo Chemical Company, Limited Production of insecticidal protein of Bacillus thuringiensis subsp. aizawai by the expression of insecticidal protein gene in host cells
US5231008A (en) * 1985-10-28 1993-07-27 Sumitomo Chemical Company, Limited Production of insecticidal protein of bacillus thuringiensis subsp. aizawal IPL by the expression of insecticidal protein gene in host cells
EP0224331A1 (de) * 1985-10-28 1987-06-03 Sumitomo Chemical Company, Limited Herstellung von Insektizidprotein von Bacillus thuringiensis subsp. aizawai IPL durch die Expression von Insektizidprotein-Gen in Wirtszellen
US5608142A (en) * 1986-12-03 1997-03-04 Agracetus, Inc. Insecticidal cotton plants
BE1001877A4 (fr) * 1988-02-25 1990-04-03 Sandoz Sa Composes nouveaux.
FR2627778A1 (fr) * 1988-02-25 1989-09-01 Sandoz Sa Gene de structure, proteine endotoxine correspondante et application insecticide
EP0366397A1 (de) * 1988-10-27 1990-05-02 Mycogen Corporation Bacillus thuringiensis, benannt B.t. ps81f, welches aktiv gegen Lepidoptera ist, sowie Gen, welches für das gegen Lepidoptera aktive Toxin kodiert
EP0367474A1 (de) * 1988-11-01 1990-05-09 Mycogen Corporation Bacillus thuringiensis, benannt B.t. ps81gg, welches aktiv gegen Lepidoptera ist, sowie Gen welches für das gegen Lepidoptera aktive Toxin kodiert

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JPS61139389A (ja) 1986-06-26
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ATE61631T1 (de) 1991-03-15
DE3582136D1 (de) 1991-04-18

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